Identification of cardiac stem cells within mature cardiac myocytes

Cardiac stem cells are described in a number of mammalian species including humans. Cardiac stem cell clusters consisting of both lineage-negative and partially committed cells are generally identified between contracting cardiac myocytes. In the present study, c-kit⁺, Sca⁺, and Isl1⁺ stem cells were revealed to be located inside the sarcoplasm of cardiac myocytes in myocardial cell cultures derived from newborn, 20-, and 40-day-old rats. Intracellularly localized cardiac stem cells had a coating or capsule with a few pores that opened into the host cell sarcoplasm. The similar structures were also identified in the suspension of freshly isolated myocardial cells (ex vivo) of 20- and 40-day-old rats. The results from this study provide direct evidence for the replicative division of encapsulated stem cells, followed by their partial cardiomyogenic differentiation. The latter is substantiated by the release of multiple transient amplifying cells following the capsule rupture. In conclusion, functional cardiac stem cells can reside not only exterior to but also within cardiomyocytes.     

Location and functional characterization of the right atrioventricular pacemaker ring in the adult avian heart

Previous histological studies showed that in addition to a sinus node, an atrioventricular (AV) node, an AV bundle, left and right bundle branches, birds also possess a right AV‐Purkinje ring that is located in the atrial sheet of the right muscular AV‐valve along all its base length. The functionality of the AV‐Purkinje ring is unknown. In this work, we studied the topology of pacemaker myocytes in the atrial side of the isolated chicken spontaneously contracting right muscular AV‐valve using the method of microelectrode mapping of action potentials. We show that AV‐cells having the ability to show pacemaking reside in the right muscular AV‐valve. Pacemaker action potentials were exclusively recorded close to the base of the valve along its whole length from dorsal to the ventral attachment to the interventricular septum. These action potentials have much slower rate of depolarization, lower amplitude, and higher diastolic depolarization than action potentials of Purkinje (conducting) cells. We conclude the right AV‐valve has a ring bundle of pacemaker cells (but not Purkinje cells) in the adult chicken heart. J. Morphol. 277:363–369, 2016. © 2015 Wiley Periodicals, Inc.     

Factors controlling embryonic heart cell proliferation in serum-free synthetic media

Summary Embryonic chick cardiac cell cultures, plated on collagen-coated dishes, containing serum-free synthetic media proliferate actively. The basic medium contained Ham's F12 nutrient mixture, fetuin, ascorbic acid, and bovine serum albumin. This medium was supplemented with various combinations of factors; endothelial cell growth supplement (ECGS), epidermal growth factor (EGF), insulin (I), transferrin (T), selenium (S), hydrocortisone, and thyroxine or supplemented alone. Basic medium supplemented with ECGS alone contributes to the highest final cell density among all other factors used in various combinations or alone. The final cell density of the control culture with 2% fetal bovine serum was higher than those of all experimental cultures and an additional control culture grown in the basic medium. Combinations of factors without ECGS do not promote significant cell proliferation. Thyroxine is required to induce optimal differentiation and contractility of cardiac myocytes in vitro. Fibronectin and laminin did not show any more influence than collagen did on the growth and maintenance of cardiac myocytes in serum-free media. The proportion of cardiac muscle cells in ECGS-containing media was higher than those in other experimental media and control media with the exception of ECGS and ITS-containing medium that showed lower proportion of cardiac myocytes than that of serum-containing medium on Days 3 and 5. The profiles of incorporation of [³H]thymidine into DNA of heart cells in experimental and control cultures showed a peak in incorporation values within the first week of culture and subsequently declined. Autoradiography studies revealed that cardiac myocytes in culture supplemented with ECGS alone attained a peak in labeling index on Day 1 with approximately 62% labeled cells. Subsequently, the labeling indices declined. Cardiac myocytes grown in media without ECGS showed significantly lower labeling indices than those in ECGS-containing media. This study has demonstrated the influence of ECGS, EGF and ITS in promoting the growth of cardiac myocytes and also in contributing to the maintenance of contractile cardiac myocytes in serum-free, long-term culture. The influence of ECGS on heart cell proliferation is considered to be superior to that of EGF and ITS. This study was supported in part by a grant HL-25482 from the National Heart Lung and Blood Institute and a grant from the American Heart Association of Michigan.     

白细胞介素-2对心肌负性肌力作用机制的探讨

为研究白细胞介素-2（IL-2）对心肌的负性肌力作用的可能机制,本采用酶解分离成年大鼠心室肌细胞,用细胞内双波长荧光系统和膜片钳全细胞记录检测细胞膜钙离子通道和细胞内酸碱度（pHi）及钙水平的变化,分别以fura-2/AM和BCECF/AM作为细胞内钙离子和氢离子荧光指示剂。结果：（1）IL-2(2.5-200U/ml）浓度依赖性地降低单个心室肌细胞电刺激诱导的钙瞬变幅度,使舒张末钙水平升高,选择性κ-阿片受体阻断剂nor-BNI(10nmol/L）可阻断IL-2对心肌细胞内钙的作用;（2）用200U/ml的IL-2灌流10min后,与对照组相比膜片钳全细胞记录的L-型钙电流无明显改变;（3）用200U/ml的IL-2灌流后,与对照组相比Mn^2 对fura-2/AM的淬灭率无明显改变。（4）IL-2(200U/ml）使大鼠心室肌细胞pHi降低,其作用可被选择性κ-阿片受体阻断剂nor-BNI(10nmol/L）所减弱。结论：IL-2引起的心室肌细胞pHi降低可能是其负性肌力作用机制之一,细胞膜上L-型钙离子通道可能不参与IL-2降低心肌收缩力的作用;细胞膜上的阿片受体可能介导IL-2对心肌的负性肌力作用。     

Role of kinases and G-proteins in the hyposmotic stimulation of cardiac IKs

Exposure of cardiac myocytes to hyposmotic solution stimulates slowly-activating delayed-rectifying K⁺ current (I_Ks) via unknown mechanisms. In the present study, I_Ks was measured in guinea-pig ventricular myocytes that were pretreated with modulators of cell signaling processes, and then exposed to hyposmotic solution. Pretreatment with compounds that (i) inhibit serine/threonine kinase activity (10-100 μM H89; 200 μM H8; 50 μM H7; 1 μM bisindolylmaleimide I; 10 μM LY294002; 50 μM PD98059), (ii) stimulate serine/threonine kinase activity (1-5 μM forskolin; 0.1 μM phorbol-12-myristate-13-acetate; 10 μM acetylcholine; 0.1 μM angiotensin II; 20 μM ATP), (iii) suppress G-protein activation (10 mM GDPβS), or (iv) disrupt the cytoskeleton (10 μM cytochalasin D), had little effect on the stimulation of I_Ks by hyposmotic solution. In marked contrast, pretreatment with tyrosine kinase inhibitor tyrphostin A25 (20 μM) strongly attenuated both the hyposmotic stimulation of I_Ks in myocytes and the hyposmotic stimulation of current in BHK cells co-expressing Ks channel subunits KCNQ1 and KCNE1. Since attenuation of hyposmotic stimulation was not observed in myocytes and cells pretreated with inactive tyrphostin A1, we conclude that TK has an important role in the response of cardiac Ks channels to hyposmotic solution.     

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca²⁺ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).¹ Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca²⁺ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca²⁺-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 μM Ca²⁺. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca²⁺ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca²⁺ and Ca²⁺ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca²⁺ transient measurements, performed in these isolated myocytes.     

Die sog. Asphalt-Flecke der kultivierten Herzmuskelzellen — eine im Phasenkontrastmikroskop sichtbare Formation des Glykogens

Zusammenfassung Bei einer korrespondierenden Betrachtung dreier Asphaltflecke von lebenden Herzmuskelzellen in der Kultur und den identischen Stellen im Elektronenmikroskop erweist sich der Fleckeninhalt als eine Anhäufung locker beieinanderliegender -Teilchen des Glykogens.Präparate für die elektronenmikroskopische Untersuchung konnten hergestellt werden, nachdem der schweren Fixierbarkeit des Fleckeninhaltes mit der Verwendung des Glutaraldehyds in Kakodylatpuffer und der Spülung mit 50%igem Alkohol nach der Reynold'schen Kontrastierung Rechnung getragen war.Mit dieser Diagnose ist zugleich bewiesen, daß Glykogen im Phasenkontrastbild der lebenden Zelle sichtbar werden kann.

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The asphalt coloured spots of living heart muscle cells — glycogen formations visible under phase contrast

Summary The contents of three asphalt coloured spots previously examined in the living heart muscle cells in a culture by a phase contrast microscope and subsequently identified in the electron microscope by the method of Gross and Riedel proved to be accumulations of -particles of glycogen loosely lying together.Suitable sections could be manufactured for electron microscope after complying with the difficulty of the spot contents to be fixated — revealed during the histochemical investigations. One had to use glutaraldehyde in cacodylate buffer as the first fixative and 50% alcohol instead of distilled water for rinsing after the Reynold's staining.By this diagnosis at the same time, it is proven that certain glycogen formations in living cells are visible under phase contrast.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Rab6A GTPase contributes to phenotypic modulation in pulmonary artery smooth muscle cells under hypoxia

Previous studies have demonstrated that hypoxia can induce phenotypic modulation of pulmonary smooth muscle cells; however, the mechanisms remain unclear. The present study aimed to investigate the effect of the GTPase Rab6A-mediated phenotypic modulation and other activities of rat pulmonary artery smooth muscle cells (RPASMCs). We revealed that Rab6A was induced by hypoxia (1% O₂) and was involved in a hypoxia-induced phenotypic switch and endoplasmic reticulum stress (ERS) in RPASMCs. After 48 hours of hypoxia, the expression of the phenotype marker protein smooth muscle actin was downregulated and vimentin (VIM) expression was upregulated. Rab6A was upregulated after 48 hours of hypoxia, and the level of glucose-regulated protein, 78 kDa (GRP78) after 12 hours of hypoxic stimulation was also increased. After transfection with a Rab6A short interfering RNA under hypoxic conditions, the expression levels of GRP78 and VIM in RPASMCs were downregulated. Overall, hypoxia-induced RPASMCs to undergo ERS followed by phenotypic transformation. Rab6A is involved in this hypoxia-induced phenotypic modulation and ERS in RPASMCs.     

In vitro cardiac performance in triploid brown trout at two acclimation temperatures

The maximum values for heart rate ( f _H), stroke volume ( V _H), cardiac output ( Q ) and myocardial power output, measured in vitro with a perfused heart preparation, as well as the isometric force-frequency relationship for atrial and ventricular muscle strips, in triploid brown trout Salmo trutta were all comparable with established information for diploid rainbow trout Oncorhynchus mykiss . Therefore, it was concluded that triploidy is not associated with a major deficiency in maximum cardiac performance. However, a heightened sensitivity to ryanodine was discovered, which indicated an enhanced role for the sarcoplasmic reticulum in excitation-contraction coupling in these triploid fish. It is suspected that the enhanced role of the ryanodine receptor may be a cellular compensation related to larger cardiac myocytes. It was also clearly established that there was a plateau in maximum cardiac performance between 14 and 18° C and this plateau might be a contributing factor to the reduced factorial aerobic scope and increased fish mortality observed at 18° C.     

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.